Method for detection of drug-induced mutations in the reverse transcriptase gene

ABSTRACT

The present invention relates to a method for the rapid and reliable detection of drug-induced mutations in the reverse transcriptase gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay. More particularly, the present invention relates to a method for determining the susceptibility to antiviral drugs of HIV strains present in a biological sample, comprising: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the reverse transcriptase genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two RT gene probes hybridizing specifically to one or more target sequences with said probes being applied to known locations on a solid support and with said probes being capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of homologous targets, or said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T is replaced by U; (iv) detecting the hybrids formed in step (iii); (v) inferring the nucleotide sequence at the codons of interest and/or the amino acids of the codons of interest and/or antiviral drug resistance spectrum, and possibly the type of HIV isolates involved from the differential hybridization signal(s) obtained in step (iv).

[0001] The present invention relates to the field of HIV diagnosis. Moreparticularly, the present invention relates to the field of diagnosingthe susceptibility of an HIV sample to antiviral drugs used to treat HIVinfection.

[0002] The present invention relates to a method for the rapid andreliable detection of drug-induced mutations in the HIV reversetranscriptase gene allowing the simultaneous characterization of a rangeof codons involved in drug resistance using specific sets of probesoptimized to function together in a reverse-hybridisation assay.

[0003] During the treatment of human immunodeficiency virus (HIV) type 1infected individuals with antiretroviral nucleoside analogs emergence ofresistance against these drugs has been observed. The mechanismresponsible for the resistance is not fully understood, since theappearance of a resistant virus in not always correlated with clinicaldeterioration (Boucher et al. 1992). Amongst the reverse transcriptase(RT) inhibitors, the nucleoside analogs 3′-azido-2′,3′-dideoxyThymidine(AZT, Zidovudine), 2′,3′-dideoxyilnosine (ddI), 2′,3′-dideoxyCytidine(ddC), (−)-β-L-2′,3′-dideoxy-3′-thioCytidine (3TC),2′,3′-didehydro-3′deoxyThymidine (D4T) and(−)-2′3′-dideoxy-5-fluoro-3′-thiacytidine (FTC) are the most important,since they show a favourable ratio of toxicity for the host versusefficacy as antiviral. All these compounds act in a similar way, namelythey serve, after intracellular phosphorylation, as chain terminators ofthe RT reaction. Upon prolonged treatment with these nucleoside analogs,accumulation of mutations in the viral reverse transcriptase gene (RT)occur, thereby escaping the inhibitory effect of the antivirals. Themost important mutations induced by the above compounds and leading togradually increasing resistance were found at amino acid (aa) positions41 (M to L), 69 (T to D), 70 (K to R), 74 (L to V), 181 (Y to C), 184 (Mto V) and 215 (T to Y or F) (Schinazi et al., 1994). Mutations at aa 65,67, 75 and 219 have also been reported but these were only showing aminor decrease in sensitivity. More recently, multi-drug-resistant HIV-1strains were described showing aa changes at codon 62, 75, 77, 116, and151 (Iversen et al., 1996). In general, these aa changes are theconsequence of single point mutations at the first or second codonletter, but in the case of T69D (ACT to GAT), T215Y (ACC to TAC) andT215F (ACC to TTC),: two nucleotide mutations are necessarry. Whether inthese cases the single nucleotide mutation intermediates exist, and ifthey show any importance in the mechanism for acquiring resistance is asyet not reported. Third letter variations are in general not leading toan amino acid change, and are therefore seen as natural polymorphisms.

[0004] The regime for an efficient antiviral treatment is not clear atall. The appearance of one or several of these mutations duringantiviral treatment need to be interpreted in conjunction with the virusload and the amount of CD4 cells. Indeed, since it has been shown thatthe effect of AZT resistance mutations can be suppressed after theappeareance of the 3TC induced M184V mutation, it is clear that diseaseprogression is multifactorial. The influence of other simultaneousoccuring mutations under different combination therapies with respect tothe outcome and resistance of the virus has not yet been analysedsystematically. In order to get a better insight into the mechanisms ofresistance and HIV biology, it is necessarry to analyse follow-up plasmasamples of antiviral treated patients for these mutational eventstogether with the simultaneous occuring changes of virus titre and CD4cells.

[0005] It is an aim of the present invention to develop a rapid andreliable detection method for determination of the antiviral drugresistance of viruses which contain reverse transcriptase genes such asHIV retroviruses and Hepadnaviridae present in a biological y sample.

[0006] More particularly it is an aim of the present invention toprovide a genotyping assay allowing the detection of the different HIVRT gene wild type and mutation codons involved in the antiviralresistance in one single experiment.

[0007] It is also an aim of the present invention to provide an HIV RTgenotyping assay or method which allows to infer the nucleotide sequenceat codons of interest and/or the amino acids at the codons of interestand/or the antiviral drug resistance spectrum, and possibly also inferthe HIV type or subtype isolate involved.

[0008] Even more particularly it is an aim of the present invention toprovide a genotyping assay allowing the detection of the different HIVRT gene polymorphisms representing wild-type and mutation codons in onesingle experimental setup.

[0009] It is another aim of the present invention to select particularprobes able to discriminate wild-type HIV RT sequences from mutated orpolymorphic HIV RT sequences conferring resistance to one or moreantiviral drugs, such as AZT, ddI, ddC, 3TC or FTC, D4T or others.

[0010] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated or polymorphic HIV RT sequences conferring resistance to AZT.

[0011] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to ddI.

[0012] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to ddC.

[0013] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to 3TC.

[0014] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to D4T.

[0015] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to FTC.

[0016] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to multiple nucleosideanalogues (i.e. multidrug resistance).

[0017] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to nevirapine.

[0018] It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HIV RT from mutated HIVRT sequences involving at least one of amino acid positions 41 (M to L),50 (I to T), 67 (D to N). 69 (T to D), 70 (K to R), 74 (L to V), 75 (Vto T), 151 (Q to M or L), 181 (Y to C), 184 (M to V). 215 (T to Y or F)and 219 (K to Q or E) of the viral reverse transcriptase (RT) gene.

[0019] It is particularly an aim of the present invention to select aparticular set of probes, able to discriminate wild-type HIV RTsequences from mutated HIV RT sequences conferring resistance to any ofthe antiviral drugs defined above with this particular set of probesbeing used in a reverse hybridisation assay.

[0020] It is moreover an aim of the present invention to combine a setof selected probes able to discriminate wild-type HIV RT sequences frommutated HIV RT sequences conferring resistance to antiviral drugs withanother set of selected probes able to identify the HIV isolate, type orsubtype present in the biological sample, whereby all probes can be usedunder the same hybridisation and wash-conditions.

[0021] It is also an aim of the present invention to select primersenabling the amplification of the gene fragment(s) determining theantiviral drug resistance trait of interest.

[0022] The present invention also aims at diagnostic kits comprisingsaid probes useful for developing such a genotyping assay.

[0023] All the aims of the present invention have been met by thefollowing specific embodiments.

[0024] The present invention relates more particularly to a method fordetermining the susceptibility to antiviral drugs of an HIV retroviruspresent in a biological sample, comprising:

[0025] (i) if need be releasing, isolating or concentrating thepolynucleic acids present in the sample;

[0026] (ii) if need be amplifying the relevant part of the reversetranscriptase genes present in said sample with at least one suitableprimer pair;

[0027] (iii) hybridizing the polynucleic acids of step (i) or (ii) withat least two RT gene probes hybridizing specifically to at least onetarget sequence as mentioned in any of FIG. 1 and tables 1, 2 or 4, withsaid probes being applied to known locations of a solid support and withsaid probes being capable of simultaneously hybridizing to theirrespective target regions under appropiate hybridization and washconditions allowing the detection of homologous targets, or with saidprobes hybridizing specifically with a sequence complementary to any ofsaid target sequences, or a sequence wherein T in said target sequenceis replaced by U;

[0028] (iv) detecting the hybrids formed in step (iii);

[0029] (v) and in most cases, inferring the nucleotide sequence at thecodons of interest and/or the amino acids at the codons of interestand/or the antiviral drug resistance spectrum, and possibly the type ofHIV isolates involved from the differential hybridization signal(s)obtained in step (iv).

[0030] The relevant part of the RT gene refers to the regions in the RTgene harboring mutations causing resistance to antiviral drugs asdescribed above and is particularly comprised between codons 1 and 241,and more particularly between codons 29 and 220 of the RT gene.

[0031] According to a preferred embodiment of the present invention,step (iii) is i performed using a set of at least 2, preferably at least3, more preferably at least 4 and most preferably at least 5 probesmeticulously designed as such that they show the desired hybridizationresults, when used in a reverse hybridisation assay format, moreparticularly under the same hybridization and wash conditions.

[0032] According to a preferred embodiment, the present inventionrelates to a set of at least 2 probes each targetting one or more of thenucleoside RT inhibitor induced nucleotide changes or target sequencesincluding such a nucleotide change as indicated in any of FIG. 1 orTables 1, 2 or 4. The numbering of HIV-1 RT gene encoded amino acids isas generally accepted in literature.

[0033] More prefererably, the present invention relates to a set of twoor more probes each targetting two, three, four, five or more differentnucleoside RT inhibitor induced nucleotide changes as indicated in anyof FIG. 1 or Tables 1, 2 or 4.

[0034] More particularly, the present invention relates to a set of atleast 2 probes allowing the characterization of a wild-type, polymorphicor mutated codon at any one of the drug-induced mutation positionsrepresented in any of FIG. 1 or Tables 1 or 2 or at any one of thepolymorphic positions represented in Table 4.

[0035] Even more particularly, the present invention relates to a set ofat least 2 probes allowing the characterization of a wild-type,polymorphic or mutated codon at any of the positions represented in FIG.1.

[0036] All the above mentioned sets of probes have as a commoncharacteristic that all the probes in said set are designed so that theycan function together in a reverse-hybridization assay, moreparticularly under similar hybridization and wash conditions.

[0037] A particularly preferred set of probes selected out of the probeswith SEQ ID NO 1 to 161 of Table 3 is described in example 2.2 and isindicated in Table 4 and FIG. 2. The particularly selected probes arealso indicated in Table 3.

[0038] A particularly preferred embodiment of the present invention is amethod for determining the susceptibility to antiviral drugs of an HIVisolates in a sample using a set of probes as defined above, whereinsaid set of probes is characterized as being chosen such that for agiven mutation disclosed in any of FIG. 1, or Tables 1, 2 or 4 thefollowing probes are included in said set

[0039] at least one probe for detecting the presence of drug inducedmutation at said position;

[0040] at least one probe for detecting the presence of a wild-typesequence at said position;

[0041] preferably also (an) additional probe(s) for detecting wild-typepolymorphisms at positions surrounding the mutation position.

[0042] Inclusion of the latter two types of probes greatly contributesto increasing the sensitivity of said assays as demonstrated in theexamples section.

[0043] A particularly preferred set of probes in this respect is shownin Tables 3 and 4 and FIGS. 2 and 3.

[0044] Selected sets of probes according to the present inventioninclude at least one probe, preferably at least two probes,characterizing the presence of a drug-induced mutation in a codonposition chosen from the following list of codons susceptible tomutations in the HIV RT gene: 41, 50, 67, 69, 70, 74, 75, 151, 181, 184,215 or 219. Said probes being characterized in that they can function ina method as set out above.

[0045] Also selected probes according to the present invention areprobes which allow to differentiate any of the nucleotide changes asrepresented in any of FIG. 1 or Tables 1, 2 or 4. Said probes beingcharacterized in that they can function in a method as set out above.

[0046] Also selected sets of probes for use in a method according to thepresent invention include at least one, preferably at least two (setsof) probes, with said probes characterizing the presence of adrug-induced mutation in two codon positions chosen from the followinglist of codon combinations, with said codons being susceptible tomutations in the HIV RT gene: 41 and/or 50; 41 and/or 67; 41 and/or 69;41 and/or 70; 41 and/or 74; 41 and/or 75; 41 and/or 151; 1 and/or 181;41 and/or 184; 41 and/or 215; 41 and/or 219; 50 and/or 67; 50 and/or 69;50 and/or 70; 50 and/or 74; 50 and/or 75; 50 and/or 75; 50 and/or 151;50 and/or 181; 50 and/or 184; 50 and/or 215; 50 and/or 219; 67 and/or69; 67 and/or 70; 67 and/or 74; 67 and/or 75; 67 and/or 151; 67 and/or181; 67 and/or 184; 67 and/or 215; 67 and/or 219; 69 and/or 70; 69and/or 74; 69 and/or 75; 69 and/or 151; 69 and/or 181; 69 and/or 184; 69and/or 215; 69 and/or 219; 70 and/or 74; 70 and/or 75; 70 and/or 151; 70and/or 181: 70 and/or 184; 70 and/or 215; 70 and/or 219: 74 and/or 75;74 and/or 151; 74 and/or 181; 74 and/or 184; 74 and/or 215; 74 and/or219; 75 and/or 151; 75 and/or 181; 75 and/or 184; 75 and/or 215; 75and/or 219; 151 and/or 181: 151 and/or 184; 151 and/or 215; 151 and/or219; 181 and/or 184; 181 and/or 215; 181 and/or 219; 184 and/or 215; 184and/or 219; 215 and/or 219.

[0047] Even more preferred selected sets of probes for use in a methodaccording to the present invention include in addition to the probesdeemed above a third (set of) probe(s) characterizing the presence of athird drug-induced mutation at any of positions 41, 50, 67, 69, 70, 74,75, 151, 181, 184, 215 or 219, or particular combinations thereof.

[0048] Particularly preferred is also a set of probes which allowssimultaneous detection of antiviral resistance at codons 41, 50, 67, 69,70, 74, 75, 151, 181, 184 and 215, possibly also at codon 219.

[0049] An additional embodiment of the present invention includes atleast one probe, preferably at least two probes, characterizing thepresence of a drug-induced mutation in codon positions chosen from thelist of codons susceptible-to mutations in the HIV RT gene as mentionedin any of Table 1 or 2, such as at codons 65, 115, 150, 98, 100, 103,106, 108, 188, 190, 138, 199, 101, 179, 236, 238 or 233, with saidprobes forming possibly part of a composition.

[0050] Particularly preferred embodiments of the invention thus includea set of probes for codon 41 comprising at least one, preferably atleast two, probe(s) for targetting at least one, preferably at leasttwo, nucleotide changes in any the following codons as represented inregion I in FIG. 1:

[0051] wild-type codon E40 (GAA) or polymorphic codon E40 (GAG), mutantcodon L41 (TTG) or L41 (CTG) or wild-type codon M41 (ATG), wild-typecodon E42 (GAA) or polymorphic codon E42 (GAG), wild-type codon K43(AAG) or polymorphic codon K43 (AAA) or polymorphic E43 (GAA).

[0052] Particularly preferred embodiments of the invention thus includea set of probes for codon 50 comprising at least one, preferably atleast two, probe(s) for targetting at least one, preferably at leasttwo, nucleotide changes in any the following codons as represented inregion II in FIG. 1:

[0053] wild-type codon K49 (AAA) or polymorphic codon R49 (AGA), mutantcodons V50 (GTT) or T50 (ACG), wild-type codon 150 (ATT) or polymorphiccodon 150 (ATC).

[0054] Particularly preferred embodiments of the invention thus includea set of probes for codons 67-70 comprising at least one, preferably atleast two, probe(s) for targetting at least one, preferably at leasttwo, nucleotide changes in any of the following codons as represented inregion III in FIG. 1:

[0055] wild-type K65 (AAA) or polymorphic K65 (AAG), wild-type K66 (AAA)or polymorphic K66 (AAG). wild-type D67 (GAC) or mutant N67 (AAC),wild-type T69 (ACT) or polymorphic T69 (ACA), mutant D69 (GAT) or N69(AAT) or A69 (GCT), wild-type K70 (AAA), polymorphic K70 (AAG) or mutantR70 (AGA).

[0056] Particularly preferred embodiments of the present inventioninclude a set of probes for codons 74-75 comprising at least one,preferably at least two, probes for targetting at least one, preferablyat least two, nucleotide chances in any of the following codons asrepresented in region IV of FIG. 1:

[0057] wild-type K73 (AAA) or polymorphic K73 (AAG), wild-type L74 (TTA)or mutant V74 (GTA), wild-type V75 (GTA) or polymorphic V75 (GTG) ormutant T75 (ACA), wild-type D76 (GAT) or polymorphic D76 (GAC).

[0058] Particularly preferred embodiments of the present inventioninclude a set of probes for codon 151 comprising at least one,preferably at least two, probes for targetting at least one, preferablyat least two, nucleotide changes in any of the following codons asrepresented in region V of FIG. 1:

[0059] wild-type L149 (CTT) or polymorphic L149 (CTC) or L149 (CTG),wild-type P150 (CCA) or polymorphic P150 (CCG), wild-type Q151 (CAG) ormutant M51 (ATG) or L151 (CTG) or polymorphic Q151 (CAA).

[0060] Particularly preferred embodiments of the present inventioninclude a set of probes for codon 181-184 comprising at least one,preferably at least two, probe(s) for targetting at least one,preferably at least two, nucleotide changes in any of the followingcodons as represented in region VI of FIG. 1:

[0061] wild-type Y181 (TAT) or mutant C181 (TGT), wild-type Q182 (CAA)or polymorphic Q182 (CAG), wild-type Y183 (TAC) or polymorphic Y183(TAT), wild-type M184 (ATG) or mutant V184 (GTG) or 1184 (ATA) or G184(AGG), wild-type D185 (GAT) or polymorphic D185 (GAC), wild-type D186(GAT) or polymorphic E186 (GAG), wild-type L187 (TTA) or polymorphicG187 (GGA) or V187 (GTA).

[0062] Particularly preferred embodiments of the present inventioninclude a set of probes for codon 215 comprising at least one,preferably at least two, probe(s) for targetting at least one,preferably at least two, nucleotide changes in any of the followingcodons as represented in region VII of FIG. 1

[0063] wild-type G213 (GGA) or polymorphic G213 (GGG), wild-type F214(TTT) or polymorphic F214 (TTC) or L214 (CTT) or L214 (TTA), wild-typeT215 (ACC) or polymorphic T215 (ACT), mutant Y215 (TAC) or F215 (TTC).

[0064] Particularly preferred embodiments of the present inventioninclude a set of probes for codon 219 comprising at least one,preferably at least two, probe(s) for targetting at least one,preferably at least two, nucleotide chances in any of the followingcodons as represented in region VIII of FIG. 1:

[0065] wild-type D218 (GAC) or polymorphic D218 (GAT), wild-type K219(AAA) or polymorphic K219 (AAG) or mutant Q219 (CAA) or E219 (GAA),wild-type K220 (AAA) or polymorphic K220 (AAG).

[0066] Examples of probes of the invention are represented in Tables 3and 4, and FIGS. 2 and 3. In Table 3, the probes withheld afterselection are indicated using the letter “y”. These probes of theinvention are designed for attaining optimal performance under the samehybridization conditions so that they can be used in sets of at least 2probes for simultaneous hybridization; this highly increases theusefulness of these probes and results in a significant gain in time andlabour. Evidently, when other hybridization conditions would bepreferred, all probes should be adapted accordingly by adding ordeleting a number of nucleotides at their extremities. It should beunderstood that these concommitant adaptations should give rise toessentially the same result, namely that the respective probes stillhybridize specifically with the defined target. Such adaptations mightalso be necessary if the amplified material should be RNA in nature andnot DNA as in the case for the NASBA (nucleic acid sequence-basedamplification) system.

[0067] The selection of the preferred probes of the present invention isbased on a reverse hybridization assay using immobilized oligonucleotideprobes present at distinct locations on a solid support. Moreparticularly the selection of preferred probes of the present inventionis based on the use of the Line Probe Assay (LiPA) principle which is areverse hybridization assay using oligonucleotide probes immobilized asparallel lines on a solid support strip (Stuyver et al. 1993;international application WO 94/12670). This approach is particularlyadvantageous since it is fast and simple to perform. The reversehybridization format and more particularly the LiPA approach has manypractical advantages as compared to other DNA techniques orhybridization formats, especially when the use of a combination ofprobes is preferable or unavoidable to obtain the relevant informationsought.

[0068] It is to be understood, however, that any other type ofhybridization assay or format using any of the selected probes asdescribed further in the invention, is also covered by the presentinvention.

[0069] The reverse hybridization approach implies that the probes areimmnobilized to certain locations on a solid support and that the targetDNA is labelled in order to enable the detection of the hybrids formed.

[0070] Methods for detecting nucleotide changes in RT genes of otherviruses which have been found to harbour a pattern of drug-resistancemutation similar to the one observed for HIV based on the sameprinciples as set out in the present invention should be understood asalso being covered by the scope of the present invention.

[0071] The following definitions serve to illustrate the terms andexpressions used in the present invention.

[0072] The term “antiviral drugs” refers particularly to an antiviralnucleoside analog or any other RT inhibitor. Examples of such antiviraldrugs and the mutation they may cause in the HIV-RT gene are disclosedin Schinazi et al., 1994 and Mellors et al., 1995. The contents of thelatter two documents particularly are to be considered as forming partof the present invention. The most important antiviral drugs focussed atin the present invention are disclosed in Tables 1 to 2.

[0073] The term “drug-induced mutation” refers to a mutation in the HIVRT gene which provokes a reduced susceptibility of the isolate to therespective drug.

[0074] The target material in the samples to be analysed may either beDNA or RNA, e.g.: genomic DNA, messenger RNA, viral RNA or amplifiedversions thereof. These molecules are also termed polynucleic acids.

[0075] It is possible to use genomic DNA or RNA molecules from HIVsamples in the methods according to the present invention.

[0076] Well-known extraction and purification procedures are availablefor the isolation of RNA or DNA from a sample (f.i. in Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarbourLaboratory Press (1989)).

[0077] The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence to be detected.

[0078] The term “target sequence” as referred to in the presentinvention describes the nucleotide sequence of the wildtype, polymorphicor drug induced variant sequence of the RT gene to be specificallydetected by a probe according to the present invention. This nucleotidesequence may encompass one or several nucleotide changes. Targetsequences may refer to single nucleotide positions, codon positions,nucleotides encoding amino acids or to sequences spanning any of theforegoing nucleotide positions. In the present invention said targetsequence often includes one or two variable nucleotide positions. It isto be understood that the complement of said target sequence is also asuitable target sequence in some cases. The target sequences as definedin the present invention provide sequences which should be complementaryto the central part of the probe which is designed to hybridizespecifically to said target region.

[0079] The term “complementary” as used herein means that the sequenceof the single stranded probe is exactly the (inverse) complement of thesequence of the single-stranded target, with the target being defined asthe sequence where the mutation to be detected is located.

[0080] Since the current application requires the detection of singlebasepair mismatches, very stringent conditions for hybridization arerequired, allowing in principle only hybridization of exactlycomplementary sequences. However, variations are possible in the lengthof the probes (see below), and it should be noted that, since thecentral part of the probe is essential for its hybridizationcharacteristics, possible deviations of the probe sequence versus thetarget sequence may be allowable towards head and tail of the probe,when longer probe sequences are used. These variations, which may beconceived from the common knowledge in the art, should however always beevaluated experimentally, in order to check if they result in equivalenthybridization characteristics than the exactly complementary probes.

[0081] Preferably, the probes of the invention are about 5 to 50nucleotides long, more preferably from about 10 to 25 nucleotides.Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides asused in the present invention may be ribonucleotides,deoxyribonucleotides and modified nucleotides such as inosine ornucleotides containing modified groups which do not essentially altertheir hybridisation characteristics.

[0082] Probe sequences are represented throughout the specification assingle stranded DNA oligonucleotides from the 5′ to the 3 end. It isobvious to the man skilled in the art that any of the below-specifiedprobes can be used as such, or in their complementary form, or in theirRNA form (wherein T is replaced by U).

[0083] The probes according to the invention can be prepared by cloningof recombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

[0084] The term “solid support” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead) or a chip. Prior to application to the membrane orfixation it may be convenient to modify the nucleic acid probe in orderto facilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

[0085] The term “labelled” refers to the use of labelled nucleic acids.Labelling may be carried out by the use of labelled nucleotidesincorporated during the polymerase step of the amplification such asillustrated by Saiki et al. (1988) or Bej et al. (1990) or labelledprimers, or by any other method known to the person skilled in the art.The nature of the label may be isotopic (32p, 35S, etc.) or non-isotopic(biotin, digoxigenin, etc.).

[0086] The term “primer” refers to a single stranded oligonucleotidesequence capable of acting as a point of initiation for synthesis of aprimer extension product which is complementary to the nucleic acidstrand to be copied. The length and the sequence of the primer must besuch that they allow to prime the synthesis of the extension products.

[0087] Preferably the primer is about 5-50 nucleotides long. Specificlength and sequence will depend on the complexity of the required DNA orRNA targets, as well as on the conditions of primer use such astemperature and ionic strenght.

[0088] The fact that amplification primers do not have to match exactlywith the corresponding template sequence to warrant proper amplificationis amply documented in the literature (Kwok et al., 1990).

[0089] The amplification method used can be either polymerase chainreaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgrenet al., 1988; Wu & Wallace, 1989; Barany, 1991), nucleic acidsequence-based amplification (NASBA; Guatelli et al., 1990; Compton,1991), transcription-based amplification system (TAS; Kwoh et al.,1989), strand displacement amplification (SDA; Duck, 1990; Walker etal., 1992) or amplification by means of Qβ replicase (Lizardi et al.,1988; Lomeli et al., 1989) or any other suitable method to amplifynucleic acid molecules known in the art.

[0090] The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids(Nielsen et al., 1991; Nielsen et al., 1993) or may containintercalating agents (Asseline et al., 1984).

[0091] As most other variations or modifications introduced into theoriginal DNA sequences of the invention these variations willnecessitate adaptions with respect to the conditions under which theoligonucleotide should be used to obtain the required specificity andsensitivity. However the eventual results of hybridisation will beessentially the same as those obtained with the unmodifiedoligonucleotides.

[0092] The introduction of these modifications may be advantageous inorder to positively influence characteristics such as hybridizationkinetics, reversibility of the hybrid-formation, biological stability ofthe oligonucleotide molecules, etc.

[0093] The “sample” may be any biological material taken either directlyfrom the infected human being (or animal), or after culturing(enrichment). Biological material may be e.g. expectorations of anykind, broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyteblood culture material, colonies, liquid cultures, faecal samples, urineetc.

[0094] The sets of probes of the present invention will include at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may beapplied in two or more distinct and known positions on a solidsubstrate. Often it is preferable to apply two or more probes togetherin one and the same position of said solid support.

[0095] For designing probes with desired characteristics, the followinguseful guidelines known to the person skilled in the art can be applied.

[0096] Because the extent and specificity of hybridization reactionssuch as those described herein are affected by a number of factors,manipulation of one or more of those factors will determine the exactsensitivity and specificity of a particular probe, whether perfectlycomplementary to its target or not. The importance and effect of variousassay conditions, explained further herein, are known to those skilledin the art.

[0097] The stability of the [probe:target] nucleic acid hybrid should bechosen to be compatible with the assay conditions. This may beaccomplished by avoiding long AT-rich sequences, by terminating thehybrids with G:C base pairs, and by designing the probe with anappropriate Tm. The beginning and end points of the probe should bechosen so that the length and %GC result in a Tm about 2-10° C. higherthan the temperature at which the final assay will be performed. Thebase composition of the probe is significant because G-C base pairsexhibit greater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

[0098] Conditions such as ionic strenght and incubation temperatureunder which a probe will be used should also be taken into account whendesigning a probe. It is known that hybridization will increase as theionic strenght of the reaction mixture increases, and that the thermalstability of the hybrids will increase with increasing ionic strenght.On the other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

[0099] It is desirable to have probes which hybridize only underconditions of high stringency. Under high stringency conditions onlyhighly comnplementary nucleic acid hybrids will form: hybrids without asufficient degree of complementarity will not form.

[0100] Accordingly, the stringency of the assay conditions determinesthe amount of complementarity needed between two nucleic acid strandsforming a hybrid. The degree of stringency is chosen such as to maximizethe difference in stability between the hybrid formed with the targetand the nontarget nucleic acid. In the present case, single base pairchanges need to be detected, which requires conditions of very highstringency.

[0101] The length of the target nucleic acid sequence and, accordingly,the length of the probe sequence can also be important. In some cases,there may be several sequences from a particular region, varying inlocation and length, which will yield probes with the desiredhybridization characteristics. In other cases, one sequence may besignificantly better than another which differs merely by a single base.While it is possible for nucleic acids that are not perfectlycomplementary to hybridize, the longest stretch of perfectlycomplementary base sequence will normally primarily determine hybridstability. While oligonucleotide probes of different lengths and basecomposition may be used, preferred oligonucleotide probes of thisinvention are between about 5 to 50 (more particularely 10-25) bases inlength and have a sufficient stretch in the sequence which is perfectlycomplementary to the target nucleic acid sequence.

[0102] Regions in the target DNA or RNA which are known to form stronginternal structures inhibitory to hybridization are less preferred.Likewise, probes with extensive self-complementarity should be avoided.As explained above, hybridization is the association of two singlestrands of complementary nucleic acids to form a hydrogen bonded doublestrand. It is implicit that if one of the two strands is wholly orpartially involved in a hybrid that it will be less able to participatein formation of a new hybrid. There can be intramolecular andintermolecular hybrids formed within the molecules of one type of probeif there is sufficient self complementarity. Such structures can beavoided through careful probe design. By designing a probe so that asubstantial portion of the sequence of interest is single stranded, therate and extent of hybridization may be greatly increased. Computerprograms are available to search for this type of interaction. However,in certain instances, it may not be possible to avoid this type ofinteraction.

[0103] Standard hybridization and wash conditions are disclosed in theMaterials & Methods section of the Examples. Other conditions are forinstance 3× SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at50° C.

[0104] Other solutions (SSPE (Sodium saline phosphate EDTA), TMACl(Tetramethyl ammonium Chloride), etc.) and temperatures can also be usedprovided that the specificity and sensitivity of the probes ismaintained. If need be, slight modifications of the probes in length orin sequence have to be carried out to maintain the specificity andsensitivity required under the given circumstances.

[0105] In a more preferential embodiment, the above-mentionedpolynucleic acids from step (i) or (ii) are hybridized with at leasttwo, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more of the above-mentioned target region specific probes,preferably with 5 or 6 probes, which, taken together, cover the“mutation region” of the RT gene.

[0106] The term “mutation region” means the region in the HIV RT genesequence where s most of the mutations responsible for antiviral drugresistance or other observed polymorphisms are located. A preferred partof this mutation region is represented in FIG. 1. This mutation regioncan be divided into 8 important parts: drug induced variations andpolymorphisms located within aa positions 38 to 44 of RT gene, druginduced variations and polymorphisms located within aa positions 47 to53 of RT gene, drug induced variations and polymorphisms located withinaa positions 65 to 72 of the RT gene, drug induced variations andpolymorphisms located within aa positions 73 to 77 of the RT gene,drug-induced variations and polymorphisms located within aa positions148 to 154 of the RT gene, drug-induced variations and polymorphismslocated within aa positions 180 to 187 of the RT gene, drug inducedvariations and polymorphisms located within aa positions 212 to 216 ofthe RT gene and drug induced variations and polymorphisms located withinaa positions 217 to 220 of the RT gene.

[0107] Since some mutations may be more frequently occurring thanothers, e.g. in certain geographic areas or in specific circumstances(e.g. rather closed communities) it may be appropiate to screen only forspecific mutations, using a selected set of probes as indicated above.This would result in a more simple test, which would cover the needsunder certain circumstances.

[0108] In order to detect the antiviral drug RT resistance pattern withthe selected set of oligonucleotide probes, any hybridization methodknown in the art can be used (conventional dot-blot, Southern blot,sandwich, etc.).

[0109] However, in order to obtain fast and easy results if a multitudeof probes are involved, a reverse hybridization format may be mostconvenient.

[0110] In a preferred embodiment the selected set of probes areimmobilized to a solid support in known distinct locations (dots, linesor other figures). In another preferred embodiment the selected set ofprobes are immobilized to a membrane strip in a line fashion. Saidprobes may be immobilized individually or as mixtures to delineatedlocations on the solid support.

[0111] A specific and very user-friendly embodiment of theabove-mentioned preferential method is the LiPA method, where theabove-mentioned set of probes is immobilized in parallel lines on amembrane, as further described in the examples.

[0112] The invention also provides for any probes and primer setsdesigned to specifically detect or amplify specifically these RT genepolymorphisms, and any method or kits using said primer and probes sets.

[0113] The invention further provides for any of the probes as describedabove, as well as compositions comprising at least one of these probes.

[0114] The invention also provides for a set of primers allowingamplification of the mutation region of the RT gene in general.

[0115] Primers may be labeled with a label of choice (e.g. biotine).Different primer-based target amplification systems may be used, andpreferably PCR-amplification, as set out in the examples. Single-roundor nested PCR may be used.

[0116] The invention also provides for a kit for inferring thenucleotide sequence at codons of interest in the HIV RT gene and/or theamino acids corresponding to these codons and/or the antiviral drugresistance spectrum of HIV isolates present in a biological samplecomprising the following components:

[0117] (i) when appropiate, a means for releasing, isolating orconcentrating the polynucleic acids present in said sample;

[0118] (ii) when appropriate, at least one of the above-defined set ofprimers;

[0119] (iii) at least two of the probes as defined above, possibly fixedto a solid support;

[0120] (iv) a hybridization buffer, or components necessary forproducing said buffer;

[0121] (v) a wash solution, or components necessary for producing saidsolution;

[0122] (vi) when appropriate, a means for detecting the hybridsresulting from the preceding hybridization.

[0123] (vii) when appropriate, a means for attaching said probe to asolid support.

[0124] The term “hybridization buffer” means a buffer enabling ahybridization reaction to occur between the probes and the polynucleicacids present in the sample, or the amplified products, under theappropiate stringency conditions.

[0125] The term “wash solution” means a solution enabling washing of thehybrids formed under the appropiate stringency conditions.

[0126] A line probe assay (LiPA) was designed for the screening forvariations at interesting amino acids in the HIV RT gene. The principleof the assay is based on reverse hybridization of an amplifiedpolynucleic acid fragment such as a biotinylated PCR fragment of the HIVRT gene onto short oligonucleotides. The latter hybrid can then, via abiotine-streptavidine coupling, be detected with a non-radioactivecolour developing system.

[0127] The present invention further relates to a reverse hybridizationmethod wherein said oligonucleotide probes are immobilized, preferablyon a membrane strip.

[0128] The present invention also relates to a composition comprisingany of the probes as defined in Tables 3 and 4 or FIGS. 2 and 3.

[0129] The present invention relates also to a kit for inferring the HIVRT resistance spectrum of HIV in a biological sample, coupled to theidentification of the HIV isolate L involved, comprising the followingcomponents:

[0130] (i) when appropiate, a means for releasing, isolating orconcentrating the polynucleic acids present in the sample;

[0131] (ii) when appropriate, at least one of the sets of primers asdefined above;

[0132] (iii) at least one of the probes as defined above, possibly fixedto a solid support;

[0133] (iv) a hybridization buffer, or components necessary forproducing said buffer;

[0134] (v) a wash solution, or components necessary for producing saidsolution;

[0135] (vi) when appropriate, a means for detecting the hybridsresulting from the preceding hybridization;

[0136] (vii) when appropriate, a means for attaching said probe to asolid support

[0137] The following examples only serve to illustrate the presentinvention. These examples are in no way intended to limit the scope ofthe present invention.

FIGURE AND TABLE LEGENDS

[0138]FIG. 1: Natural and drug induced variability in the vicinity ofcodons 41, 50, 67-70, 74-75, 150, 181-184, 215 and 219 of the HIV RTgene. The most frequently observed wild-type sequence is shown in thetop line. Naturally occuring variations are indicated below.Drug-induced variants are indicated in bold italics

[0139]FIG. 2A. Reactivities of the selected probes for codon 41immobilized on LiPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number given each time. *:False positive reactivities. On top of the strips, the amino acids atthe relevant codon, as derived from the probe reactivity, is indicated.

[0140]FIG. 2B. Reactivities of the selected probes for codons 69-70immobilized on LIPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given ID Ad inTable 4. Each strip is incubated with a biotinylated PCR fragment fromthe reference panel. The reference panel accession numbers are indicatedin Table 4. For several probes multiple reference panel possibilitiesare available, but only one relevant accession number given each time.*: False positive reactivities. On top of the strips, the amino acids atthe relevant codon, as derived from the probe reactivity, is indicated.

[0141]FIG. 2C. Reactivities of the selected probes for codons 74-75immobilized on LiPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number given each time. Ontop of the strips, the amino acids at the relevant codon, as derivedfrom the probe reactivity, is indicated.

[0142]FIG. 2D. Reactivities of the selected probes for codon 184immobilized on LiPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number given each time. Ontop of the strips, the amino acids at the relevant codon, as derivedfrom the probe reactivity, is indicated.

[0143]FIG. 2E. Reactivities of the selected probes for codon 215immobilized on LIPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number W given each time. Ontop of the strips, the amino acids at the relevant codon, as derivedfrom the probe reactivity, is indicated.

[0144]FIG. 2F. Reactivities of the selected probes for codon 219immobilized on LIPA strips with reference material. The position of eachprobe on the membrane strip is shown at the right of each panel. Thesequence of the relevant part of the selected probes is given in Table4. Each strip is incubated with a biotinylated PCR fragment from thereference panel. The reference panel accession numbers are indicated inTable 4. For several probes multiple reference panel possibilities areavailable, but only one relevant accession number given each time. Ontop of the strips, the amino acids at the relevant codon, as derivedfrom the probe reactivity, is indicated.

[0145]FIG. 3. Clinical and virological features detectable in threepatient follow-up samples. All three patients were infected with a HIV-1strain showing the M41-T69-K70-L74-V75-M184-F214-T215-K219 genotype(wild type pattern). Top: Fluctuations between plasma HIV RNA copynumbers (▪) and CD4 cell count (x) are given in function of time. Thedifferent treatment regimens and the period of treatment is indicated ontop. Middle: Changes that appeared during the treatment period and thatcould be scored by means of the LiPA probes are indicated, for patient91007 at codon 41 and 215; for patient 94013 at codon 184; for patient92021 at codon 70, 214, 215, 219. Bottem: Corresponding LiPA strips fora subset of the aa changes are shown. LiPA probes are indicated on theleft, the aa interpretation is indicated at the right of each panel.

[0146]FIG. 4. Reactivities of the selected probes for codons 151 and 181on LiPA strips with reference material. The position of each probe onthe membrane strip is shown at the right of each panel. The sequence ofthe relevant part of the selected probes is given in Table 3. LIPAstrips were incubated with sequence-confirmed PCR fragments, extractedand amplified from: a wild-type HIV-1 isolate (strip 1), a wild-typeisolate with a polymorphism at codon 151 (strip 2) or 149 (strip 3), amulti-drug resistant HIV-1 isolate (strip 4) with no information aboutcodon 181 and a non-nucleoside analogue treated HIV-1 isolate whichremained wild-type at codon 151 (strip5).

[0147] Table 1: Mutations in HIV-1 RT gene associated with resistanceagainst nucleoside RT inhibitors. More details are given in Mellors etal., 1995.

[0148] Table 2: Mutations in HIV-1 RT gene associated with resistanceagainst HIV-1 specific RT inhibitors. For more details see Mellors-etal., 1995.

[0149] Abbreviations in Table 1 and 2:

[0150] AZT: 3′-azido-2′3′-dideoxythymidine

[0151] ddC: 2′3′-dideoxycytidine

[0152] ddI: 2′3′-dideoxyinosine

[0153] 3TC: 3′dideoxy-3′-thiacytidine

[0154] FTC: 2′3′-dideoxy-5-fluoro-3′-thiacytidine

[0155] L′697,593:5-ethyl-6-methyl-3-(2-phthalimido-ethyl)pyridin-2(1H)-one

[0156] L′697.661: 3-Il(4,7-dichloro-1,3-benzoxazol-2-yl)methylamino-5-ethyl-6-methylpyridin-2(1H)-one

[0157] Nevirapine:1l-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyridol(3,2-b:2′,3′-e)diazepin-6-one

[0158] TIBO R82150:(+)-(5S)-4,5,6,7,-tetrahydro-5-methyl-6-(3-methyl-2butenyl)imidazo(4,5,1-)k)(1,4)-benzodiazepin-2(1H)-thione

[0159] TIBO 82913:(+)-(5S)-4,5,6,7,-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1kj)-(1,4)benzo-diazepin-2(1H)-thione

[0160] TSAO-m²T :(2′,5′-bis-o-(tert-buthyldimethylsilyl)-3′-spiro-5′-(4′-amino-1′,2′-oxathiole-2′,2′-dioxide)

[0161] U90152:1-(3-(1-methylethyl)-amino)-2-pyridinyl)4-(5-(methylsulphonyl)-amino)-1H-indol-2yl)-carbonyl)-piperazine

[0162] Table 3: HIV RT wild-type and drug resistance mutation probes.The probes witheld after selection are indicated as “y”.

[0163] Table 4: Prediction and prevalence of LIPA probe reactivity.Probe names corresponding with the selected motifs are presented in theleft column, with the relevant part of each probe shown under theconsensus. The prevalence of these motives, determined using panels ofEuropean and US sera, is given in the middle column. The right columnindicates the corresponding strips of FIGS. 2A-F and the accessionnumber of the reference panel clone used to obtain this reactivity.

EXAMPLES Example 1

[0164] a. Materials and Methods.

[0165] Plasma samples were taken from HIV type-1 infected patients andstored at −20° C. until use. Patients were treated with AZT, ddI, ddC,D4T, 3TC, or several combinations of these prodrugs. The European serumsamples tested were randomly selected. For the US serum collection, onlythe first sample from a follow-up series was taken. Some of these USpatients were treated, others were not treated.

[0166] HIV RNA was prepared using the guanidinium-phenol procedure.Fifty μl plasma was mixed with 150 μl Trizol® LS Reagent (LifeTechnologies, Gent, Belgium) at room temperature (volume ratio: 1 unitsample/3 units Trizol). Lysis and denaturation occured by carefullypipetting up and down several times, followed by an incubation step atroom temperature for at least 5 minutes. Fourty μl CHCl₃ was added andthe mixture was shaken vigorously by hand for at least 15 seconds, andincubated for 15 minutes at room temperature. The samples werecentrifuged at maximum 12,000 g for 15 minutes at 4° C., and thecolourless aquous phase was collected and mixed with 100 μl isopropanol.To visualize the minute amounts of viral RNA, 20 μl of 1 μg/μl DextranT500 (Pharmacia) was added, mixed and left at room temperature for 10minutes. Following centrifugation at max. 12,000 g for 10 minutes at 4°C. and aspiration of the supernatant, the RNA pellet was washed with 200μl ethanol, mixed by vortexing and collected by centrifugation at 7,500g for 5 minutes at 4° C. Finally the RNA pellet was briefly air-dryedand stored at −20° C.

[0167] For cDNA synthesis and PCR amplification, the RNA pellet wasdissolved in 15 μl random primers (20 ng/μl, pdN₆, Pharmacia), preparedin DEPC-treated or HPLC grade water. After denaturation at 70° C. for 10minutes, 5 μl cDNA mix was added, composed of 4 μl 5× AMV-RT buffer (250mM Tris.HCl pH 8.5, 100 mM KCl, 30 mM MgCl₂, 25 mM DTT), 0.4 μL 25 mMdXTPs, 0.2 μl or 25U Ribonuclease Inhibitor (HPRI, Amersham), and 0.3 μlor 8U AMV-RT (Stratagene). cDNA synthesis occured during the 90 minutesincubation at 42° C. The HIV RT gene was than amplified using thefollowing reaction mixture: 5 μl cDNA, 4.5 μl 10× Taq buffer, 0.3 μl 25mM dXTPs, 1 μl (10 pmol) of each PCR primer, 38 μl H₂O, and 0.2 μl (1 U)Taq. The primers for amplification had the following sequence: outersense RT-9: 5′ bio-GTACAGTATTAGTAGGACCTACACCTGTC 3′(SEQ ID NO 96);nested sense RT-1: 5′ bio-CCAAAAGTTAAACAATGGCCATTGACAGA 3′ (SEQ ID NO97); nested antisense RT-4: 5′ bio-AGTTCATAACCCATCCAAAG 3′ (SEQ ID NO98); and outer antisense primer RT-12: 5′bio-ATCAGGATGGAGTTCATAACCCATCCA 3′ (SEQ ID NO 99). Annealing occured at57° C., extension at 72° C. and denaturation at 94° C. Each step of thecycle took 1 minute, the outer PCR contained 40 cycles, the nested round35. Nested round PCR products were analysed on agarose gel and onlyclearly visible amplification products were used in the LIPA procedure.Quantification of viral RNA was obtained with the HIV Monitor™test(Roche, Brussels, Belgium).

[0168] Selected PCR products, amplified without 5′ biotine primers, werecloned into the pretreated EcoRV site of the pGEMT vector (Promega).Recombinant clones were selected after α-complementation and restrictionfragment length analysis, and sequenced with plasmid primers andinternal HIV RT primers. Other biotinylated fragments were directlysequenced with a dye-terminator protocol (Applied Biosystems) using theamplification primers. Alternatively, nested PCR was carried out withanalogs of the RT-4 and RT-1 primers, in which the biotine group wasreplaced with the T7- and SP6-primer sequence, respectively. Theseamplicons were than sequenced with an SP6- and 17-dye-primer procedure.Sequence information was submitted to the GENBANK.

[0169] Probes were designed to cover the different polymorphisms anddrug induced mutations. In principle, only probes that discriminatedbetween one single nucleotide variation were retained. However, forcertain polymorphisms at the extreme ends of the the probe,cross-reactivity was tolerated. Specificity was reached for each probeindividually after considering the % (G+C), the probe lenght, the finalconcentration of the buffer components, and hybridization temperature.Optimized probes were provided enzymatically with a poly-T-tail usingthe TdT (Pharmacia) in a standard reaction condition, and purified viaprecipitation. Probe pellets were disolved in standard saline citrate(SSC) buffer and applied as horizontal parallel lines on a membranestrip. Control lines for amplification (probe 5′CCACAGGGATGGAAAG 3′, HIVRT aa 150 to aa 155) and conjugate incubation (biotinylated DNA) wereapplied alongside. After fixation of the probes onto the membranes bybaking, membranes were sliced into 4 mm strips.

[0170] To perform LiPA tests, equal amounts (10 μl) of biotinylatedamplification products and denaturation mixture (0.4 N NaOH/0.1% SDS)were mixed, followed by an incubation at room temperature for 5 minutes.Following this denaturation step, 2 ml hybridization buffer (2× SSC,0.1% SDS, 50 mM Tris pH 7.5) was added together with a membrane stripand hybridization was carried out at 39° C. for 30 min. Then, thehybridization mixture was replaced by stringent washing buffer (samecomposition as hybridisation buffer), and stringent washing occuredfirst at room temperature for 5 minutes and than at 39° C. for another25 minutes. Buffers were than replaced to be suitable for thestreptavidine alkaline phosphatase conjugate incubations. After 30minutes incubation at room temperature, conjugate was rinsed away andreplaced by the substrate components for alkaline phosphatase,Nitro-Blue-Tetrazolium and 5-Bromo-4-Chloro-3-Indolyl Phosphate. After30 minutes incubation at room temperature, probes where hybridizationoccured became visible because of the purple brown precipitate at thesepositions.

[0171] b. Results.

[0172] b.1 The HIV-1 RT Gene PCR and Selection of a Reference Panel.

[0173] PCR primers were chosen outside the target regions for probedesign. The amplified region located inside the nested primers coveredthe HIV-1 RT gene from codon 29 to codon 220. The primer design wasbased on published sequences from the HIV-1 genotype B clade. Europeanand United States HIV-1 positive serum samples, stored appropriately (at−20° C.) without repeating freezing-thawing cycles, were PCR positive in96% of the cases (not shown). The annealing temperature for the selectedprimers seemed to be crucial (57° C.). At 55° C., a second aspecificamplicon of approximately 1500-bp was generated; and at 59° C. theamount of specific fragment decreased drastically. With the currentprimer combination, the corresponding RT region could be amplified fromisolates of the genotype A, C, D and F clade, but with a reducedsensitivity.

[0174] A total of 25 selected PCR fragments with the targetpolymorphisms and mutations were retained as reference panel andsequenced on both strands. The selection occurred during the evaluationof the probes, and these samples originated from naive or drug-treatedEuropean or US patients. Biotinylated PCR products from this panel(Accession Number L78133 to L78157) were used to test probes forspecificity and sensitivity.

[0175] b.2 Nucleotide Target Region for Probe Design and ProbeSelection.

[0176] Table 4 and parts of FIG. 1 are a compilation of the natural anddrug-selected variability in the vicinity of aa 41, 69-70, 74-75, 184,215, and 219 of the HIV RT gene. To create this table and parts of thisfigure, the “National Centre for Biotechnology Information” database wassearched and all HIV-1 genome entries were retrieved and analyzed one byone. Only those entries displaying non-ambiguous sequence information inthe vicinity of the above-mentioned codons were retained for furtherinterpretation. It should be noted that the indicated variations do notimply that they occur in the same sequence: for example the variabilityobserved at codon 40 and 43 may occasionally occur together, but mostoften, if they occur, only one of them is found. In these 6 regions, atotal of 19 different third-letter and two first-letter (codon 43 AAGversus GAG and codon 214 TTT versus CTT) polymorphisms need to beincluded in the selection of wild type probes. Another 13 first-letterand/or second-letter variations are drug-induced and are the maintargets for the selection of probes (FIG. 1).

[0177] For the design of relevant probes, only those database motifsthat systematically returned (highly prevalent motif) were included,while scattered mutations which were found randomly (low prevalentmotif, not shown) were ignored. Based on database sequences, sevenmotifs for codon 41 (91.6% of all entrees), 6 for codon 69-70 (86.2%), 2for codon 74-75 (90.4%), 5 for codon 184 (96.6%), 9 for codon 215(94.1%), and 2 for codon 219 (88.2%) were selected (Table 4).

[0178] Probe names corresponding with the selected motifs are presentedin the left column of Table 4, with the relevant sequence part of eachprobe shown under the consensus. The prevalence of these motives wasthan determined using panels of European and US sera (Table 4).

[0179] In many cases, the database entries were not representative forthe samples tested.

[0180] Upon analyzing the European and US samples, many were notreactive with these database-selected probes. Upon sequencing analysisof several of these unreactive PCR products, another 8 motifs becameapparent, for which the corresponding probes were designed (41w20,41m12, 70 m13, 74w9, 74m6, 74 m12, 184w24, 215m49). By including thesenewly designed motifs, negative results were markedly decreased; for allcodon positions except codon 219, the total percentage of reactivityexceeds 90%.

[0181] Another 4 probes were designed (41 m11, 215m50, 219m7, and 219m9)because their sequence motif was found in the cloned reference panel,although no reactivity with the tested plasma virus samples was found sofar. The existence of these rare sequence motifs is explained byassuming that they exist at an extremely low frequency in the viralquasispecies, remaining undetectable by direct detection methods, butbecoming apparent after cloning.

[0182] The sequence motif of probe 215m13 was generated in recombinantclones by site-directed mutagenesis (not shown). The rational behindthis probe design was to determine whether the sequence combination ofcodon Y215 (TAC) can occur in combination with L214 (CT) in vivo.However, this latter motif was not found in the plasma samples tested.

[0183] Four probes (41w15, 70w8, 215w29, 215w27) are in fact redundant,because they detect identical sequence motifs covered by other probes.However, the location of these redundant probes is slightly different totheir sequence-identical counterpart. These probes have the potential toavoid negative results which might otherwise appear as a consequence ofrandom mutations in the probe target area and can therfore increase thespecificity of recognition.

[0184] b.3 Probe Specificity and Sensitivity.

[0185] The 48 selected probes were applied separately on LIPA strips.Biotinylated PCR fragments generated from the reference panel ordirectly from plasma virus were alkali-denatured, the hybridizationbuffer and LiPA strips were added, and submitted to stringenthybridization and washing conditions. Positions where hybridizationoccurred were revealed by the biotine-streptavidine calorimetricdetection system. FIGS. 2 (A to F) shows the reactivity of these 48designed probes. In the right columns of Table 4, there is theindication of the corresponding strip in FIG. 2, and the accessionnumber of the reference panel clone used to obtain this reactivity. Thereactivities of these probes were concordant with the nucleotidesequences. False positive reactivities were observed only for probe41w19 (FIG. 2A.9) and for 70m3 (FIG. 2B.8), with extremely rare sequencemotifs 41m12 (prevalence less than 0.3%) and 70m16 (not experimentallyfound), respectively. Weak cross-reactivity, as was observed on probe41m13 with a 41m27 motif (FIG. 2A.10) was, in general, not tolerated inthe probe design. When occurring, however, it never influenced thegenotypic resistance interpretation.

[0186] b.4 Applicability of the LiPA in Patient Management.

[0187] We selected follow-up samples from three patients and analyzedthe viral genotype on the 48 LIPA probes. FIG. 3 illustrates theapplicability of genotypic resistance measurement in conjunction withthe analysis of viral load and CD4 cell count. All three patients had awild type virus (i.e. M41-T69-K70-L74-V75-M184-F214-T219-K219) strain inthe sample collected before anti-retroviral treatment. Codon positionsthat changed upon treatment are presented in FIG. 3.

[0188] From Patient 91007, 11 serum samples were analyzed, the firstsample being collected 2 weeks before the start of therapy. The LIPArevealed that before treatment, in a T215 context, two variants at codonposition 213 were predominantly present (GGG and GGA respectivelydetected by probe 215w11 and 215w9/215w29). From week 50 until week 81,a mixture of T215 and Y215 could be detected. Both variants at codon 213were also represented in the selected resistant genotypes (probes 215m17and 215m14 are positive). From week 94 onwards, only Y215 mutant viruscould be detected. A nearly identical geno-conversion at codon 41 wasobserved, with the detection of mixtures (M41 and L41) from week 81until week 111; from week 126 onwards, only L41 could be found (stripsnot shown). CD4 values were highly variable. Nevertheless, a continuousdecrease in CD4 is apparent (p=0.019, linear regression analysis). Viralload also decreased initially. However, the direct response to thetreatment might have been missed in this follow-up series, since thefirst sample after the start of the treatment is at 32 weeks. From thanon, viral load increased.

[0189] Patient 94013 was treated with 3TC monotherapy from week 2onwards. At week 10, a mixture of M184 and V184 could be detected. Fromweek 14 on, only V184 was present. CD4 counts increased nearly 2.5-fold,with the highest level at week 10. Viral load decreased spectacularly by3 log units. But from week 10 onwards, a slight but steady increase toweek 23 was noted. The decrease in CD4 and increase in viral loadcoincided with the appearance of the V184 motif.

[0190] Patient 92021 was followed for 55 weeks. AZT treatment started atweek 10, followed by a supplemental ddC treatment from week 20 onwards.The first sample was found to be reactive with probe 215w9/w29(F214T215=TTTACC), but trace amounts of reactivity with 215w53(L214T215=TTAACC) could be detected as well, indicating the presence ofat least two variants at that time. From week 19 onwards, the codonL214=TTA motif became more important. At week 42, the first sign ofgenotypic resistance could be detected by the presence of a F214Y215motif (TTTTAT). Finally at week 55, only F214Y215 could be detected. TheL214=TTA motif disappeared completely. At week 42, a mixture (K and R)at codon 70 was present, but at week 55, only R70 could be detected. Atweek 55, a mixture of codon 219 motifs (K and E) was found (strips notshown). CD4 initially increased, with a maximal effect during AZTmonotherapy peaking at week 21. From then on, a continuous decrease wasobserved. However, ten weeks of AZT treament did not result in a drop inviral load, since the values of week 16 and 19 were nearly unchanged. Itis only after start of the combination therapy (week 20) that the viralload dropped by 1.67 log. From this patient, it is tempting to assumethat L214T215 confers genotypic resistance to AZT treatment, and thatthe addition of ddC is necessary to induce the natural F214Y215genotype. The rise in CD4 cell count may be the consequence of the drugitself, and not from drug-induced protection (Levy et al. 1996).

[0191] c. Discussion

[0192] By adapting the previously designed LiPA technology (Stuyver etal. 1993) for the HIV RT gene, the described assay format permits therapid and simultaneous detection of wild type and drug-selected variantsassociated with the genotypic resistance for AZT, ddI, ddC, d4T, FTC and3TC. The Inno LiPA HIV drug-resistance strip provides information aboutthe genetic constitution of the RT gene in the vicinity of codon 41, 69,70, 74, 75, 184, 215, and 219 at the nucleotide and, hence, also at thededuced protein level. Essentially, the biotinylated RT PCR product ishybridized against immobilized specific oligonucleotides (Table 4),which are directed against the indicated codon variabilities. Followingthis reverse-hybridization, the oligonucleotide-biotinylated-PCR-strandhybrid is recognized by the streptavidine-alkaline phosphate conjugate,which then in turn converts the alkaline phosphate substrate into apurple brown precipitate.

[0193] Using this assay, we studied the specificity and reactivity of 48probes, covering 6 different regions. This combination should allow thereliable detection of most of the genetic resistance-related codoncombinations observed to date. Occasionally occurring mutations in thevicinity of the target codons, not taken into consideration during probedesign, may eventually prevent hybridization of the probes for aparticular target region. This problem is partially solved by theredundancy of probes at the most important codons. Results obtainedusing 358 HIV infected plasma samples showed that, depending on thecodon position under investigation, between 82.4% and 100% of thecombinations could be detected, or an average of 92.7%. It is importantto mention here that the assay was developed for resistence detection ofthe HIV-1 genotype B, and only limited information is currentlyavailable about the outcome of this assay with other genotypes. Sincethe amplification primer combination is more or less universal for allthe HIV-1 isolates, some of the indeterminate results may well be due tothe presence of non-genotype B virus strains.

[0194] So far, several assays for the detection of the wild-type anddrug-selected mutations in the HIV RT gene have been described. Theseinclude Southern blotting (Richman et al., 1991), primer-specific PCR(Larder et al., 1991), PCR-LDR (Frenkel et al., 1995), RNAse A mismatchcleaving (Galandez-Lopez et al., 1991), and hybridization againstenzyme-labeled probes (Eastman et al., 1995). The general advantage ofthe LiPA and other genotypic assays is the speed by which results areobtained when compared to phenotypic assays. The particular advantage ofour test is its multi-parameter (in this particular case multi-codon)format. Moreover, the assay can easily be extended not only for thescreening of the other RT-codons, but also for proteinase codonsassociated with resistance (Mellors et al., 1995). As was illustrated inFIG. 3, mixtures of wild-type and drug-selected mutations can bedetected easily. The detection limit for these mixtures is dependent onthe sensitivity of the probes, but reliable results can be obtained assoon as 5 to 10% of the minor component is present (not shown). We wereunable to provide reliable evidence for mixtures with any sequencingprotocol at the same sensitivity level.

[0195] Due to the large amount of variables that need to be included inthe selection of specific probes (temperature of hybridization, ionicstrength of hybridization buffer, length of the probe, G+C content,strand polarity), it might occasionally occur that some of the probeswill show weak false positive reaction with related but hithertounreported sequences. In our experience, and if this occurred, this hasnever influenced the interpretation at the deduced aa level. In thecurrent selection of probes, all except two (41w19 and 70m3) wereretained on the basis of 100% specificity: as soon as one nucleotidediffers in the probe area, hybridization is abolished. Furtherfine-tuning of these two probes will therefore be necessary to obtainthe required specificity.

[0196] Accompanying polymorphisms in the vicinity of the target codonsare found with a rather high prevalence in wild-type virus strains, butnot in mutant sequences. A partial list of such combinations is herebypresented: codon V74=GTA without polymorphism at codon 73, 75 or 76;codon V184=GTG without codon Q182=CAG; and codon F215=TTC withoutF214=TTC/TTA or L214=CTT. The most intriguing example is the following:L214T215 (CTTACC) is predicted for approximately 7.8% of the wild typesequences. The corresponding motif L214Y215 (CTTTAT) apparently does notexist in plasma virus. From the example shown in FIG. 3, it is clearthat selection of mutants is a very flexible and complex phenomena. Inthis particular case, viruses having codon F214 were replaced by a L214viral population in the AZT monotherapy period, but upon selecting forgenotypic drug resistance at codon 215, the original F214 configurationwas restored. Clearly, the selection for the Y215 genotype prohibits thepresence of a L214 genotype. Since no evidence has yet emerged that L214confers resistance to anti-retroviral compounds, the appearance of thisspecial mutant during the AZT monotherapy period is difficult tointerpret. More research will certainly be necessary to clarify thisissue. But if L214 should indeed provide low-level genotypic resistanceto AZT treatment, approximately 7.8% of the naive infections will notbenefit from initial AZT monotherapy.

[0197] Since antiviral treatment can result in a marked extension oflife expectancy for HIV infected patients, it is of utmost importance tofind the best drug regimen for each individual separately. Therefore,monitoring of the magnitude and duration of the virus load and CD4 cellchanges is a prerequisite. However, knowledge concerning the geneticconstitution of the virus may also be an important factor in designingoptimal treatment schedules. Optimizing therapies making good use ofavailable information (viral load, CD4 cell count, genetic resistance)has remained largely unexploited. If this was partially due to thecomplexity of screening for all the mutational events, theabove-described LiPA technology should remove one key obstacle.

[0198] In conclusion, we have described a genotypic assay for thedetection of wild type and drug selected codons in the HIV RT gene. Thecombination of the assay result along with viral load and CD4 cellmonitoring should permit better design of patient-dependent optimaltreatment schedules.

Example 2

[0199] Multi-Drug Resistant (MDR) HIV-1 isolates have been described.These MDR isolates are characterized by having mutations in theirgenome, compared to the wild type HIV-1 genome, which result in a set ofamino acid changes. A key mutation leading to multi-drug resistance wasfound to be localized in codon 151 of the HIV-1 RT gene. Consequently,and as detecting these MDR isolates is clinically important, we designedprobes recognizing wild-type (probe 151w2) and mutant (probes 151m4 and151 m19) HIV-1 isolates. Furthermore, the presence of polymorphisms inthe direct vicinity of codon 151 (codon 149) and at codon 151 have beendescribed. Therefore, we also designed two additional probes (probes151w6 and 151w11) which detect these polymorphisms (FIG. 4 and Table 3).

[0200] Treatment with non-nucleoside analogues, such as Nevirapine(Boehringer Ingelheim), selects for several amino acid changes inconserved regions of the HIV-1 RT gene. One of the most important aminoacid changes is Y181C, a codon change that confers high levelresistance. As the detection of this mutation is also clinicallyimportant, we designed probes recognizing the wild-type (181w3 and181w5) and mutant (181m7) isolates (FIG. 4 and Table 3).

[0201]FIG. 4 shows the application of the selected probes for codon 151and 181. The position of the probes on the strips is indicated on theright side of the strips. LiPA strips were incubated withsequence-confirmed PCR fragments, extracted and amplified from: a wildtype HIV-1 isolate (strip 1), a wild type HIV-1 isolate with apolymorphism at codon 151 (strip 2) or codon 149 (strip 3), a multi-drugresistant HIV-1 isolate (strip 4) with no information about codon 181and a non-nucleoside analogue-treated HIV-1 isolate which remained wildtype at codon 151(strip 5). TABLE 1 AZT M41L ATG to TTG or CTG D67N GAGto AAC K70R AAA to AGA T215Y ACC to TAC T215F ACC to TTC K219Q AAA toCAA K219E AAA to GAA ddl K65R AAA to AGA L74V TTA to GTA V75T GTA to ACAM184V ATG to GTG ddC K65R AAA to AGA T69D ACT to GAT L74V TTA to GTAV75T GTA to ACA M184V ATG to GTG Y215C TTC to TGC d4T I50T ATT to ACTV75T GTA to ACA 3TC or FTC M184V ATG to GTG or GTA M1841 ATG to ATA1592U89 K65R AAA to AGA L74V TTA to GTA Y115F TAT to TTT M184V ATG toGTG

[0202] TABLE 2 Nevirapine A98G GCA to GGA L100I TTA to ATA K103N AAA toAAC V106A GTA to GCA V108I GTA to ATA Y181C TAT to TGT Y181I TGT to ATTY188C TAT to TGT G190A GGA to GCA TIBO L1991 TTA to ATA R82150 TIBOL100I TTA to ATA R82913 K103N AAA to AAC V106A GTA to GCA E138K GAG toAAG Y181C TAT to TGT Y188H TAT to CAT Y188L TAT to TTA L697,593 K103NAAA to AAC Y181C TAT to TGT L697,661 A98G GCA to GGA L100I TTA to ATAL697,661 K101E AAA to GAA K103N AAA to AAC K103Q AAA to CAA V108I GTA toGCA V179D GTT to GAT V179E GTT to GAG Y181C TAT to TGT BHAP U-90152P236L CCT to CTT BHAP K101E AAA to GAA U-87201 K103N AAA to AAC Y181CTAT to TGT Y188H TAT to CAT E233V GAA to GTA P236L CCT to CTT K238T AAAto ACA BHAP L100I TTA to ATA U-88204 V106A GTA to GCA Y181C TAT to TGTY181I TGT to ATT HEPT Y188C TAT to TGT E-EBU Y181C TAT to TGT E-EBU-dMY106A GTA to GCA E-EPU and Y181C TAT to TGT E-EPSeU Y188C TAT to TGTa-APA Y181C TAT to TGT R18893 S-2720 G190E GGA to GAA TSAO E138K GAG toAAG BM + 51.0836 Y181C TAT to TGT

[0203] TABLE 3 HIV RT wild-type and drug resistance SEQ ID NO PROBEFormula probe Sequentie oligo selection wild-type probes for positionM41 E40M41K43 41w7 AGAAATGGAAAAGGA   1 y E40M41K43 41w15 TGTACAGAAATGGAA  2 y M41K43 41w16 AAATGGAAAAGGAAG  3 E40M41 41w18 TACAGAGATGGAAA  4E40M41K43 41w19 GTACAGAGATGGAAA  5 E40M41K43 41w20 AGAGATGGAAAAAGA   6 yE40M41K43 41w30 AGAAATGGAGAAGGA   7 y E40M41 41w31 ACAGAGATGGAAAA  8E40M41 41w32 GTACAGAGATGGAA   9 y E40M41K43 41w33 CAGAGATGGAAAAG  10E40M41K43 41w34 AGAAATGGAAAAAGA  11 E40M41K43 41w35 GAAATGGAAAAAGA  12E40M41K43 41w36 CAGAAATGGAAAAAGA  13 y E40M41K43 41w37 AGAAATGGAAAAAGAA 14 drug-induced variant probes for position L41 E40L41K43 41m8AGAATTGGAAAAGGA  15 E40L41K43 41m11 AGAGTTGGAAAAGGA  16 y E40L41K4341m12 AGAGCTGGAAAAGG  17 y E40L41K43 41m13 AGAACTGGAAAAGG  18 yE40L41K43 41m14 GAGCTGGAAAAGG  19 E40L41K43 41m21 ACAGAATTGGAAAAG  20 yE40L41 41m22 ACAGAATTGGAAAA  21 E40L41 41m23 ACAGAACTGGAAAA  22E40L41K43 41m24 AGAATTGGAAGAGG  23 y E40L41E43 41m25 CAGAATTGGAAGAGG  24E40L41E43 41m26 AGAATTGGAAGAGGA  25 E40L41E43 41m27 AGAACTGGAAGAGG  26 yE40L41E43 41m28 CAGAACTGGAAGAGG  27 E40L41E43 41m29 AGAACTGGAAGAGGA  28wild-type probes forpositions I50 or V50 or T50 K49I50 50w4CAAAAATTGGGCCT  29 y R49I50 50w9 ATTTCAAGAATTGGG  30 y K49V50 50w5TTCAAAAGTTGGGC  31 y K49I50 50w13 CAAAAATCGGGCCTG  32 y K49T50 50w14AAAAATCGGGCCTGA  33 y wild-type probe for position D67 K64K65K66D67 67w4AAAGAAGAAAGACAG  34 y drug-induced variant probe for position N67K64K65K66N67 67m19 ATAAAGAAAAAGAACAGTA  35 y wild-type probes forpositions T69 or K70 T69K70 70w1 AGTACTAAATGGAGAA  36 y D69K70 70w2AGTGATAAATGGAGAA  37 y T69K70 70w8 ACAGTACTAAATGGAG  38 y K70K73 70w11TAAATGGAGAAAAITAG  40 drug-induced variant probes for positions D69 orN69 or A69 or R70 D69R70 70m3 GTGATAGATGGAGAA  41 T69R70 70m6GTACTAGATGGAGA  42 T69R70 70m12 AGTACTAGATGGAGA  43 y T69R70 70m13AGTACAAGATGGAGA  44 y N69R70 70m14 CAGTAATAGATGGAG  45 y A69R70 70m15ACAGTGCTAGATGGA  46 A69R70 70m16 CAGTGCTAGATGGA  47 y A69R70 70m17CAGTGCTAGATGGA  48 D69R70 70m18 CAGTGATAGATGGA  49 y D69R70 70m19CAGTGATAGATGGAG  50 D69R70 70m20 AGTGATAGATGGAG  51 D69R70 70m21AGTGATAGATGGAGA  52 wild-type probes for positions L74 or V75K73L74V75D76 74w5 GAGAAAATTAGTAGATTT  53 y K73L74V75D76 74w8AAAATTAGTAGACTTC  54 y K73L74V75D76 74w9 GAGAAAGTTAGTGGATT  55drug-induced variant probes for positions V74 or T75 K73V74V75D76 74m6AGAAAAGTAGTAGATTT  56 y K73L74T75D76 74m10 AAAATTAACAGATTTC  57K73L74T75D76 74m11 GAAAATTAACAGATTT  58 K73L74T75D76 74m12GAAAATTAACAGATTTC  59 y wild-type probes for position Q151 P150Q151G152151w2 CTTCCACAGGGATGG  60 y P150Q151G152 151w6 CTTCCACAAGGATGG  61 yP150Q151G152 151w11 TGCTCCCACAGGGATG  62 y drug-induced variant probefor position M151 P150M151G152 151m4 CTTCCAATGGGATGG  63 y P150M151G152151m19 GCTTCCAATGGGATGG  64 y wild-type probe for position Y181 Y181181w3 AGTTATCTATCAATACAG  65 y drug-induced variant probe for positionC181 C181 181m7 AGTTATCTGTCAATAC  66 y wild-type probes for positionM184 Q182M184 184w11 TCAATACATGGATGAGG  67 y Q182M184 184w17TCAGTACATGGATGAGG  68 y Q182M184 184w18 ATCAATACATGGATGA  69 Q182M184184w19 TCAGTACATGGATG  70 Q182M184 184w21 ATCAATATATGGATG  71 y Q182M184184w22 ATCAATATATGGATGA  72 Q182M184 184w23 TCAATATATGGATGA  73 Q182M184184w24 TCAATACATGGACGA  74 y Q182M184 184w25 CAATACATGGACGAT  75Q182M184 184w26 TCAATACATGGACGAT  76 drug-induced variant probes forposition V184 or I184 Q182V184 184m12 CAATACGTGGATGAGGG  77 y I184184m13 AATACATAGATGAT  78 Q182I184 184m14 CAATACATAGATGAT  79 Q182I184184m15 CAATACATAGATGATT  80 Q182V184 184m16 CAATACGTAGATGAT  81 Q182V184184m20 TCAATACGTGGATGA  82 Q182I184 184m27 TCAATACATAGATGAT  83 Q182I184184m28 ATCAATACATAGATGAT  84 y wild-type probes for position T215G213F214T215 215w9 GGATTTACCACACCA  85 y L214T215 215w10 GACTTACCACACCA 86 y F214T215 215w11 GGTTTACCACACCA  87 y F214T215 215w16GATTTACCACACCA  88 T215 215w22 TTACTACACCAGAC  89 y T215 215w24TTACCACACCAGA  90 G213L214T215 215w27 TGGGGACTTACCAC  91 y G213F214T215215w29 TGGGGATTTACCAC  92 y G213F214T215 215w32 GGGGTTCACCACAC  93G213F214T215 215w33 GGGATTCACCACAC  94 y G213F214T215 215w34GGGATTTACCACACCAG  95 G213L214T215 215w35 TGGGGACTTACCACACC  96G213F214T215 215w36 TGGGGGTTTACCACACC  97 G213F214T215 215w37GGGATTTACTACACCAG  98 G213L214T215 215w52 GGGATTAACCACAC  99G213L214T215 215w53 GGGGATTAACCACA  100 y G213L214T215 215w54TGGGGATTAACCACA 101 G213L214T215 215w55 GGGGGTTAACCACA 102 G213L214T215215w56 GGGGTTAACCACAC 103 G213L214T215 215w57 TGGGGGTTAACCAC 104G213L214T215 215w65 GGGATTGACCACAC 105 G213L214T215 215w66GGATTGACCACACC 106 G213L214T215 215w67 GGGATTGACCACA  107 y G213L214T215215w68 GGGACTGACCACA  108 y G213L214T215 215w69 GGGACTGACCACAC 109G213L214T215 215w70 TGGGGGTTAACCACA 110 G213L214T215 215w71TGTGGTTAACCCCCA  111 y G213L214T215 215w51 GGGGCTTACCACAC 112drug-induced variant probes for position Y215 or F215 G213L214Y215215m13 GGACTTTACACACC  113 y G213F214Y215 215m14 GGGTTTTACACACC  114 yG213F214F215 215m15 GGATTTTTCACACCA 115 G213F214Y215 215m17GGATTTTACACACC  116 y G213F214Y215 215m38 GGGATTTTACACACCAG 117G213F214F215 215m39 GGGATTTTTCACACCAG 118 G213F214Y215 215m40GGGATTTTACACAC 119 G213F214Y215 215m41 GGGGATTTTACACA 120 G213F214Y215215m43 CCCTAAAATGTGTG 121 G213F214F215 215m44 GGATTTTTCACACC 122F214F215 215m45 GATTTTTCACACCA  123 y G213F214F215 215m46 GGGATTTTTCACAC124 G213F214Y215 215m42 CCCCTAAAATGTGT 125 F214Y215 215m47GGTTTTATACACCA 126 G213F214Y215 215m48 GGGTTTTATACACC 127 G213F214Y215215m49 GGGGTTTTATACAC  128 y G213L214T215 215m50 GGGGGCTTACCACA  129 yG213F214Y215 215m61 GGATTCTACACACC  130 y F214Y215 215m62 GATTCTACACACC131 G213F214Y215 215m63 GGATTCTACACAC 132 G213F214Y215 215m64GGGATTCTACACAC 133 G213F214Y215 215m72 GGGTTTTATACCCC 134 F214Y215215m73 GGTTTTATACCCC 135 F214Y215 215m74 GTTTTATACCCCA 136 wild-typeprobes for position K219 K219 219w1 ACCAGACAAAAAACA 137 K219 219w2ACCAGACAAAAAAC  138 y K219 219w3 CACCAGACAAAAAAC 139 K219 219w13CAGACAAGAAACAT 140 K219 219w14 CCAGACAAGAAACA 141 K219 219w15ACCAGACAAGAAACA 142 K219 219w16 AGACAAAAAGCATC  143 y K219 219w17CAGACAAAAAGCAT 144 K219 219w18 CAGACAAAAAGCATC 145 K219 219w19CCAGATAAAAAACA 146 K219 219w20 ACCAGATAAAAAAC 147 K219 219w21CCCAGATAAAAAACA 148 K219 219w22 CCAGATAAAAAACATC 149 K219 219w23CACCAGATAAAAAAC 150 K219 219w24 CAGACAAGAAACATC 151 K219 219w25ACCAGACAAGAAAC 152 drug-induced variant probes for position Q219 or E219Q219 219m4 ACCAGACCAAAAACA 153 E219 219m5 ACCAGACGAAAAACA 154 Q219 219m6ACCAGATCAAAAACA 155 Q219 219m7 ACCAGATCAAAAAC  156 y Q219 219m8CACCAGATCAAAAAC 157 E219 219m9 ACCAGACGAAAAAC  158 y E219 219m10CCAGACGAAAAACA 159 Q219 219m11 CCAGACCAAAAACA 160 Q219 219m12ACCAGACCAAAAAC 161

[0204] TABLE 4 Prediction and prevalence of LiPA prohe reactivityconsensus prevalence Corresponding nucleic acid amino acid databaseEurope US Rp Figure probe Codon 38-43 n = 191/m = 25 n = 306 n = 52 n =25 strip Acc. Nb TGTACAGAAATGGAAAAG CTEMEK 41w7       ------------  ---- 122 (62.9%) 237 35 11  1a.1 L78149 41w15* --------------- -----118 230 38 9 1a.1 L78149 41w19    -----G------  ----  5 (2.6%) 10 2 21a.2 L78156 41w20       --G--------A   ----  0 6 0 1 1a.3 L78157 41w30      --------G---   ----  1 (0.5%) 8 6 1 1a.4 L78154 41m21   ------T--------  --L--  18 (9.4%) 37 7 2 1a.5 L78136 41m11      --GT--------   -L--  0 0 0 1 1a.6 L78140 41m24       ---T-----G--  -L-E  12 (6.3%) 1 2 1 1a.7 L78144 41m13       ---C--------   -L--  14(7.3%) 21 3 1 1a.8 L78139 41m12       --GC--------   -L--  0 1 0 1 1a.9L78155 41m27       ---C-----G--   -L-E  3 (1.6%) 0 1 1 1a.10 L78137total 175 (91.6%) 95.1% 100% 88% probe Codon 68-72 n = 354/m= 32 n = 306n = 52 n = 25 AGTACTAAATGGAGA STKWR 70w1 --------------- ----- 224(63.3%) 230 39 13 1b.1,2 L78147 70w8* ------------ ---- 208 210 38 111b.2 L78144 70m12 -------G------- --R--  37 (10.5%) 46 6 4 1b.3 L7814870m13 -----A-G------- --R--  0 0 1 2 1b.4 L78133 70w2 ---GA-----------D---  25 (7.1%) 4 4 2 1b.5 L78136 70m3    GA--G-------  DR--  10 (2.8%)3 1 0 1b.6 pending 70m14 ------------ -NR-  7 (2.0%) 4 5 2 1b.7 L7815470m16 ---G---G---- -AR-  2 (0.6%) 0 0 1 1b.8 L78150 total 305 (86.2%)91.8% 94.2% 96% probe Codon 72-77 n = 364/m = 20 n = 306 n = 52 n = 25AGAAAATTAGTAGATTTC RKLVDF 74w5 --------------- ----- 320 (87.9%) 264 4816 1c.1 L78150 74w8    -----------C---  -----  9 (2.5%) 34 1 2 1c.2L78147 74w9 -----G-----G--- -----  0 17 3 2 1c.3 L78137 74m6------G-------- --V--  0 5 0 3 1c.4 L78149 74m12    ------AC----  --T-- 0 1 1 1 1c.5 L78136 total 329 (90.4%) 93.5% 98.1% 96% probe Codon182-185 n = 322/m = 12 n = 306 n = 52 n = 25 CAATACATGGAT QYMD 184w11------------ ---- 285 (88.5%) 267 46 18 1d.1 L78147 184w17 --G-------------  16 (5.0%) 9 4 3 1d.2 L78137 184w21 -----T------ ----  6 (1.9%) 4 21 1d.3 L78145 184w24 -----------C ----  0 1 0 1 1d.4 L78144 184m12------G----- --V-  1 (0.3%) 8 0 1 1d.5 L78142 184m28 --------A--- --T- 3 (0.9%) 0 0 1 1d.6 L78148 total 311 (96.6%) 93.8 98.1% 100% probeCodon 212-218 n = 321/m = 36 n = 306 n = 52 n = 25 TGGGGATTTACCACACCAGACWGFTTPD 215w11      G------------   ----  9 (2.8%) 15 3 2 1e.1 L78146215w9    ---------------  ----- 142 (44.2%) 178 24 3 1e.2 L78141215w29^(f) ------------ ---- 142 105 16 3 1e.2 L78141 215w33   -----C------  ----  9 (2.8%) 8 4 1 1e.3 L78154 215w10      C-----------   L---  25 (7.8%) 10 0 2 1e.4 L78150 215w27^(f)------C----- --L-  25 14 0 2 1e.4 L78150 215m50    --GC--------  -L--  00 0 1 1e.5 L78145 215w53    -----A------  -L--  1 (0.3%) 1 3 1 1e.6L78138 215w22          --T---------    ----  3 (0.9%) 10 2 1 1e.7 L78134215m17 ------TA----  --Y-  88 (27.4%) 50 12 7 1e.8 L78144 215m14   --G---TA----  --Y-  24 (7.5%) 24 1 1 1e.9 L78149 215m49   --G---TAT---  --Y-  0 2 0 2 1e.10 L78148 215m45       ---TT------  -F--  1 (0.3%) 16 0 1 1e.11 L78135 215m13    ---C--TA----  -LY--  0 00 2 1e.12 L78155 total 302 (94.1%) 92.8% 90.4% 96% probe Codon 217-220 n= 204/m = 12 n = 34 n = 52 n = 26 CCAGACAAAAAA PDKK 219w2 ---------------- 179 (87.7%) 26 42 18 1f.1 L78144 219m4 ------C----- --Q- 1 (0.5%) 24 2 1f.2 L78135 219m7 -----TC----- --Q- 0 0 0 1 1f.3 L78133 219m9------G----- --E- 0 0 0 1 1f.4 pending total 179 (88.2%) 82.4% 82.7%84.6%

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1 164 1 15 DNA Artificial sequence Synthetic Primer 1 agaaatggaa aagga15 2 15 DNA Artificial sequence Synthetic Primer 2 tgtacagaaa tggaa 15 315 DNA Artificial sequence Synthetic Primer 3 aaatggaaaa ggaag 15 4 14DNA Artificial sequence Synthetic Primer 4 tacagagatg gaaa 14 5 15 DNAArtificial sequence Synthetic Primer 5 gtacagagat ggaaa 15 6 15 DNAArtificial sequence Synthetic Primer 6 agagatggaa aaaga 15 7 15 DNAArtificial sequence Synthetic Primer 7 agaaatggag aagga 15 8 14 DNAArtificial sequence Synthetic Primer 8 acagagatgg aaaa 14 9 14 DNAArtificial sequence Synthetic Primer 9 gtacagagat ggaa 14 10 14 DNAArtificial sequence Synthetic Primer 10 cagagatgga aaag 14 11 15 DNAArtificial sequence Synthetic Primer 11 agaaatggaa aaaga 15 12 14 DNAArtificial sequence Synthetic Primer 12 gaaatggaaa aaga 14 13 16 DNAArtificial sequence Synthetic Primer 13 cagaaatgga aaaaga 16 14 16 DNAArtificial sequence Synthetic Primer 14 agaaatggaa aaagaa 16 15 15 DNAArtificial sequence Synthetic Primer 15 agaattggaa aagga 15 16 15 DNAArtificial sequence Synthetic Primer 16 agagttggaa aagga 15 17 14 DNAArtificial sequence Synthetic Primer 17 agagctggaa aagg 14 18 14 DNAArtificial sequence Synthetic Primer 18 agaactggaa aagg 14 19 13 DNAArtificial sequence Synthetic Primer 19 gagctggaaa agg 13 20 15 DNAArtificial sequence Synthetic Primer 20 acagaattgg aaaag 15 21 14 DNAArtificial sequence Synthetic Primer 21 acagaattgg aaaa 14 22 14 DNAArtificial sequence Synthetic Primer 22 acagaactgg aaaa 14 23 14 DNAArtificial sequence Synthetic Primer 23 agaattggaa gagg 14 24 15 DNAArtificial sequence Synthetic Primer 24 cagaattgga agagg 15 25 15 DNAArtificial sequence Synthetic Primer 25 agaattggaa gagga 15 26 14 DNAArtificial sequence Synthetic Primer 26 agaactggaa gagg 14 27 15 DNAArtificial sequence Synthetic Primer 27 cagaactgga agagg 15 28 15 DNAArtificial sequence Synthetic Primer 28 agaactggaa gagga 15 29 14 DNAArtificial sequence Synthetic Primer 29 caaaaattgg gcct 14 30 15 DNAArtificial sequence Synthetic Primer 30 atttcaagaa ttggg 15 31 14 DNAArtificial sequence Synthetic Primer 31 ttcaaaagtt gggc 14 32 15 DNAArtificial sequence Synthetic Primer 32 caaaaatcgg gcctg 15 33 15 DNAArtificial sequence Synthetic Primer 33 aaaaatcggg cctga 15 34 15 DNAArtificial sequence Synthetic Primer 34 aaagaagaaa gacag 15 35 19 DNAArtificial sequence Synthetic Primer 35 ataaagaaaa agaacagta 19 36 16DNA Artificial sequence Synthetic Primer 36 agtactaaat ggagaa 16 37 16DNA Artificial sequence Synthetic Primer 37 agtgataaat ggagaa 16 38 16DNA Artificial sequence Synthetic Primer 38 acagtactaa atggag 16 39 27DNA Artificial sequence Synthetic Primer 39 atcaggatgg agttcataacccatcca 27 40 16 DNA Artificial sequence Synthetic Primer 40 taaatggagaaaatag 16 41 15 DNA Artificial sequence Synthetic Primer 41 gtgatagatggagaa 15 42 14 DNA Artificial sequence Synthetic Primer 42 gtactagatggaga 14 43 15 DNA Artificial sequence Synthetic Primer 43 agtactagatggaga 15 44 15 DNA Artificial sequence Synthetic Primer 44 cagtaatagatggag 15 45 15 DNA Artificial sequence Synthetic Primer 45 cagtaatagatggag 15 46 15 DNA Artificial sequence Synthetic Primer 46 acagtgctagatgga 15 47 14 DNA Artificial sequence Synthetic Primer 47 cagtgctagatgga 14 48 14 DNA Artificial sequence Synthetic Primer 48 cagtgctagatgga 14 49 14 DNA Artificial sequence Synthetic Primer 49 cagtgatagatgga 14 50 15 DNA Artificial sequence Synthetic Primer 50 cagtgatagatggag 15 51 14 DNA Artificial sequence Synthetic Primer 51 agtgatagatggag 14 52 15 DNA Artificial sequence Synthetic Primer 52 agtgatagatggaga 15 53 18 DNA Artificial sequence Synthetic Primer 53 gagaaaattagtagattt 18 54 16 DNA Artificial sequence Synthetic Primer 54 aaaattagtagacttc 16 55 17 DNA Artificial sequence Synthetic Primer 55 gagaaagttagtggatt 17 56 17 DNA Artificial sequence Synthetic Primer 56 agaaaagtagtagattt 17 57 16 DNA Artificial sequence Synthetic Primer 57 aaaattaacagatttc 16 58 16 DNA Artificial sequence Synthetic Primer 58 gaaaattaacagattt 16 59 17 DNA Artificial sequence Synthetic Primer 59 gaaaattaacagatttc 17 60 15 DNA Artificial sequence Synthetic Primer 60 cttccacagggatgg 15 61 15 DNA Artificial sequence Synthetic Primer 61 cttccacaaggatgg 15 62 16 DNA Artificial sequence Synthetic Primer 62 tgctcccacagggatg 16 63 15 DNA Artificial sequence Synthetic Primer 63 cttccaatgggatgg 15 64 16 DNA Artificial sequence Synthetic Primer 64 gcttccaatgggatgg 16 65 18 DNA Artificial sequence Synthetic Primer 65 agttatctatcaatacag 18 66 16 DNA Artificial sequence Synthetic Primer 66 agttatctgtcaatac 16 67 17 DNA Artificial sequence Synthetic Primer 67 tcaatacatggatgagg 17 68 17 DNA Artificial sequence Synthetic Primer 68 tcagtacatggatgagg 17 69 16 DNA Artificial sequence Synthetic Primer 69 atcaatacatggatga 16 70 14 DNA Artificial sequence Synthetic Primer 70 tcagtacatggatg 14 71 15 DNA Artificial sequence Synthetic Primer 71 atcaatatatggatg 15 72 16 DNA Artificial sequence Synthetic Primer 72 atcaatatatggatga 16 73 15 DNA Artificial sequence Synthetic Primer 73 tcaatatatggatga 15 74 15 DNA Artificial sequence Synthetic Primer 74 tcaatacatggacga 15 75 15 DNA Artificial sequence Synthetic Primer 75 caatacatggacgat 15 76 16 DNA Artificial sequence Synthetic Primer 76 tcaatacatggacgat 16 77 17 DNA Artificial sequence Synthetic Primer 77 caatacgtggatgaggg 17 78 14 DNA Artificial sequence Synthetic Primer 78 aatacatagatgat 14 79 15 DNA Artificial sequence Synthetic Primer 79 caatacatagatgat 15 80 16 DNA Artificial sequence Synthetic Primer 80 caatacatagatgatt 16 81 15 DNA Artificial sequence Synthetic Primer 81 caatacgtagatgat 15 82 15 DNA Artificial sequence Synthetic Primer 82 tcaatacgtggatga 15 83 16 DNA Artificial sequence Synthetic Primer 83 tcaatacatagatgat 16 84 17 DNA Artificial sequence Synthetic Primer 84 atcaatacatagatgat 17 85 15 DNA Artificial sequence Synthetic Primer 85 ggatttaccacacca 15 86 14 DNA Artificial sequence Synthetic Primer 86 gacttaccacacca 14 87 14 DNA Artificial sequence Synthetic Primer 87 ggtttaccacacca 14 88 14 DNA Artificial sequence Synthetic Primer 88 gatttaccacacca 14 89 14 DNA Artificial sequence Synthetic Primer 89 ttactacaccagac 14 90 13 DNA Artificial sequence Synthetic Primer 90 ttaccacacc aga13 91 14 DNA Artificial sequence Synthetic Primer 91 tggggactta ccac 1492 14 DNA Artificial sequence Synthetic Primer 92 tggggattta ccac 14 9314 DNA Artificial sequence Synthetic Primer 93 ggggttcacc acac 14 94 17DNA Artificial sequence Synthetic Primer 94 gggatttacc acaccag 17 95 17DNA Artificial sequence Synthetic Primer 95 gggatttacc acaccag 17 96 17DNA Artificial sequence Synthetic Primer 96 tggggactta ccacacc 17 97 17DNA Artificial sequence Synthetic Primer 97 tgggggttta ccacacc 17 98 17DNA Artificial sequence Synthetic Primer 98 gggatttact acaccag 17 99 14DNA Artificial sequence Synthetic Primer 99 gggattaacc acac 14 100 14DNA Artificial sequence Synthetic Primer 100 ggggattaac caca 14 101 15DNA Artificial sequence Synthetic Primer 101 tggggattaa ccaca 15 102 14DNA Artificial sequence Synthetic Primer 102 gggggttaac caca 14 103 14DNA Artificial sequence Synthetic Primer 103 ggggttaacc acac 14 104 14DNA Artificial sequence Synthetic Primer 104 tgggggttaa ccac 14 105 14DNA Artificial sequence Synthetic Primer 105 gggattgacc acac 14 106 14DNA Artificial sequence Synthetic Primer 106 ggattgacca cacc 14 107 13DNA Artificial sequence Synthetic Primer 107 gggattgacc aca 13 108 13DNA Artificial sequence Synthetic Primer 108 gggactgacc aca 13 109 14DNA Artificial sequence Synthetic Primer 109 gggactgacc acac 14 110 15DNA Artificial sequence Synthetic Primer 110 tgggggttaa ccaca 15 111 15DNA Artificial sequence Synthetic Primer 111 tgtggttaac cccca 15 112 14DNA Artificial sequence Synthetic Primer 112 ggggcttacc acac 14 113 14DNA Artificial sequence Synthetic Primer 113 ggactttaca cacc 14 114 14DNA Artificial sequence Synthetic Primer 114 gggttttaca cacc 14 115 15DNA Artificial sequence Synthetic Primer 115 ggatttttca cacca 15 116 14DNA Artificial sequence Synthetic Primer 116 ggattttaca cacc 14 117 17DNA Artificial sequence Synthetic Primer 117 gggattttac acaccag 17 11817 DNA Artificial sequence Synthetic Primer 118 gggatttttc acaccag 17119 14 DNA Artificial sequence Synthetic Primer 119 gggattttac acac 14120 14 DNA Artificial sequence Synthetic Primer 120 ggggatttta caca 14121 14 DNA Artificial sequence Synthetic Primer 121 ccctaaaatg tgtg 14122 14 DNA Artificial sequence Synthetic Primer 122 ggatttttca cacc 14123 14 DNA Artificial sequence Synthetic Primer 123 gatttttcac acca 14124 14 DNA Artificial sequence Synthetic Primer 124 gggatttttc acac 14125 14 DNA Artificial sequence Synthetic Primer 125 cccctaaaat gtgt 14126 14 DNA Artificial sequence Synthetic Primer 126 ggttttatac acca 14127 14 DNA Artificial sequence Synthetic Primer 127 gggttttata cacc 14128 14 DNA Artificial sequence Synthetic Primer 128 ggggttttat acac 14129 14 DNA Artificial sequence Synthetic Primer 129 gggggcttac caca 14130 14 DNA Artificial sequence Synthetic Primer 130 ggattctaca cacc 14131 13 DNA Artificial sequence Synthetic Primer 131 gattctacac acc 13132 13 DNA Artificial sequence Synthetic Primer 132 ggattctaca cac 13133 14 DNA Artificial sequence Synthetic Primer 133 gggattctac acac 14134 14 DNA Artificial sequence Synthetic Primer 134 gggttttata cccc 14135 13 DNA Artificial sequence Synthetic Primer 135 ggttttatac ccc 13136 13 DNA Artificial sequence Synthetic Primer 136 gttttatacc cca 13137 15 DNA Artificial sequence Synthetic Primer 137 accagacaaa aaaca 15138 14 DNA Artificial sequence Synthetic Primer 138 gggactgacc acac 14139 15 DNA Artificial sequence Synthetic Primer 139 caccagacaa aaaac 15140 14 DNA Artificial sequence Synthetic Primer 140 cagacaagaa acat 14141 14 DNA Artificial sequence Synthetic Primer 141 ccagacaaga aaca 14142 15 DNA Artificial sequence Synthetic Primer 142 accagacaag aaaca 15143 14 DNA Artificial sequence Synthetic Primer 143 agacaaaaag catc 14144 14 DNA Artificial sequence Synthetic Primer 144 cagacaaaaa gcat 14145 15 DNA Artificial sequence Synthetic Primer 145 cagacaaaaa gcatc 15146 14 DNA Artificial sequence Synthetic Primer 146 ccagataaaa aaca 14147 14 DNA Artificial sequence Synthetic Primer 147 accagataaa aaac 14148 15 DNA Artificial sequence Synthetic Primer 148 cccagataaa aaaca 15149 16 DNA Artificial sequence Synthetic Primer 149 ccagataaaa aacatc 16150 15 DNA Artificial sequence Synthetic Primer 150 caccagataa aaaac 15151 15 DNA Artificial sequence Synthetic Primer 151 cagacaagaa acatc 15152 14 DNA Artificial sequence Synthetic Primer 152 accagacaag aaac 14153 15 DNA Artificial sequence Synthetic Primer 153 accagaccaa aaaca 15154 15 DNA Artificial sequence Synthetic Primer 154 accagacgaa aaaca 15155 15 DNA Artificial sequence Synthetic Primer 155 accagatcaa aaaca 15156 14 DNA Artificial sequence Synthetic Primer 156 accagatcaa aaac 14157 15 DNA Artificial sequence Synthetic Primer 157 caccagatca aaaac 15158 14 DNA Artificial sequence Synthetic Primer 158 accagacgaa aaac 14159 14 DNA Artificial sequence Synthetic Primer 159 ccagacgaaa aaca 14160 14 DNA Artificial sequence Synthetic Primer 160 ccagaccaaa aaca 14161 14 DNA Artificial sequence Synthetic Primer 161 accagaccaa aaac 14162 29 DNA Artificial sequence Synthetic Primer 162 gtacagtattagtaggacct acacctgtc 29 163 29 DNA Artificial sequence Synthetic Primer163 ccaaaagtta aacaatggcc attgacaga 29 164 20 DNA Artificial sequenceSynthetic Primer 164 agttcataac ccatccaaag 20

1. Method for determining the susceptibility to antiviral drugs ofviruses which contain reverse transcriptase genes and are present in abiological sample, comprising: (i) if need be releasing, isolating orconcentrating the polynucleic acids present in the sample; (ii) if needbe amplifying the relevant part of the reverse transcriptase genespresent in said sample with at least one suitable primer pair; (iii)hybridizing the polynucleic acids of step (i) or (ii) with at least twoRT gene probes, with said probes being applied to known locations on asolid support and with said probes being capable of simultaneouslyhybridizing to their respective target regions under appropiatehybridization and wash conditions allowing the detection of homologoustargets, or with said probes hybridizing specifically with a sequencecomplementary to any of said target sequences, or a sequence wherein Tof said target sequence is replaced by U; (iv) detecting the hybridsformed in step (iii); (v) inferring the nucleotide sequence at thecodons of intrest as represented in any of FIG. 1, or Tables 1, 2 or 4and/or the amino acids of the codons of intrest and/or antiviral drugresistance spectrum, and possibly the type of viral isolates involvedfrom the differential hybridization signal(s) obtained in step (iv). 2.Method according to claim 1, wherein said viruses are HIV strains. 3.Method according to claim 2, wherein said RT gene probes hybridizespecifically to one or more target sequences as represented in any ofFIG. 1 or tables 1, 2 or
 4. 4. Method according to claim 1, wherein step(iii) consists of hybridizing with at least two probes hybridizingspecifically to one or more target codons within region I as representedin FIG.
 1. 5. Method according to claim 1, wherein step (iii) consistsof hybridizing with at least two probes hybridizing specifically to oneor more target codon within region II as represented in FIG.
 1. 6.Method according to claim 1, wherein step (iii) consists of hybridizingwith at least two probes hybridizing specifically to one or more targetcodons within region III as represented in FIG.
 1. 7. Method accordingto claim 1, wherein step (iii) consists of hybridizing with at least twoprobes hybridizing specifically to one or more target codons withinregion IV as represented in FIG.
 1. 8. Method according to claim 1,wherein step (iii) consists of hybridizing with at least two probeshybridizing specifically to one or more target codons within region V asrepresented in FIG.
 1. 9. Method according to claim 1, wherein step(iii) consists of hybridizing with at least two probes hybridizingspecifically to one or more target codons within region VI asrepresented in FIG.
 1. 10. Method according to claim 1, wherein step(iii) consists of hybridizing with at least two probes hybridizingspecifically to one or more target codons within region VII asrepresented in FIG.
 1. 11. Method according to claim 1, wherein step(iii) consists of hybridizing with at least two probes hybridizingspecifically to one or more target codons within region VII asrepresented in FIG.
 1. 12. Method according to claim 1, wherein step(iii) consists of hybridizing with at least one first probe hybridizingspecifically to one or more target codons within any region I to VII asrepresented in FIG. 1 and at least one other second probe hybridizingspecifically to one or more target codons within any region I to VIII asrepresented in FIG.
 1. 13. Probe on a solid support which is suitablefor hybridizing in a method as defined in any of claims 1 to 12 andwhich is preferably represented in Table
 3. 14. Composition comprisingat least two probes according to claim
 12. 15. A kit for inferring thenucleotide sequence at codons of interest in the HIV RT gene and/or theamino acids corresponding to these codons and/or the antiviral drugresistance spectrum of HIV isolates present in a biological samplecomprising the following components: (i) when appropiate, a means forreleasing, isolating or concentrating the polynucleic acids present insaid sample; (ii) when appropriate, at least one of the above-definedset of primers; (iii) at least two of the probes as defined above,possibly fixed to a solid support; (iv) a hybridization buffer, orcomponents necessary for producing said buffer; (v) a wash solution, orcomponents necessary for producing said solution; (vi) when appropriate,a means for detecting the hybrids resulting from the precedinghybridization. (vii) when appropriate, a means for attaching said probeto a solid support.
 16. A kit for inferring the HIV RT resistancespectrum of HIV in a biological sample, coupled to the identification ofthe HIV isolate involved, comprising the following components: (i) whenappropiate, a means for releasing, isolating or concentrating thepolynucleic acids present in the sample; (ii) when appropriate, at leastone of the sets of primers as defined above; (iii) at least one of theprobes as defined above, possibly fixed to a solid support; (iv) ahybridization buffer, or components necessary for producing said buffer;(v) a wash solution, or components necessary for producing saidsolution; (vi) when appropriate, a means for detecting the hybridsresulting from the preceding hybridization; (vii) when appropriate, ameans for attaching said probe to a solid support.